O carrapato bovino Rhipicephalus (Boophilus) microplus é o principal vetor da riquétsia Anaplasma marginale, o agente etiológico da anaplasmose, uma doença que acomete os rebanhos e causa sérios prejuízos econômicos à pecuária no Brasil. Estabelecemos em nosso laboratório o cultivo da linhagem de células BME26 que são originárias do R. (B.) microplus e também a infecção dessas células por A. marginale, um patógeno que é naturalmente transmitido pelo carrapato. Detectamos que a expressão gênica da defensina e da ixodidina nas células é aumentada frente à infecção por A. marginale, embora nenhuma alteração da expressão gênica da microplusina foi constatada. As células foram expostas a microorganismos inativados por calor e LPS, sendo que a expressão gênica da microplusina é aumentada frente a todos os estímulos. Na exposição das células BME26 com a bactéria Microccocus luteus, a expressão gênica da defensina e da ixodidina não foi alterada e no estimulo com leveduras a expressão gênica da ixodidina foi reprimida. Frente à infecção por A. marginale detectamos, aumento expressão da defensina e ixodidina. Os genes da microplusina e defensina foram silenciadas por RNAi em células infectadas por A. marginale, mas não houve alteração no número de riquétsias / The cattle tick Rhipicephalus (Boophilus) microplus is the main vector of the rickettsia Anaplasma marginale, the etiological agent of anaplasmosis, a disease that affects cattle and causes serious economic losses to the Brazilian cattle industry. We established in our laboratory the embryonic cell culture line BME26 from R. (B.) microplus and infection by A. marginale, a pathogen naturally transmitted by R. (B.) microplus. We verified that defensin and ixodidin gene expression increased in these cells after an infection by A. marginale and no alteration in microplusin gene expression was detected. The BME26 cells were exposed to heat-inactivated microorganims or to LPS, microplusin gene expression increased after all stimuli. After exposure of BME26 cells to Micrococcus luteus, expression levels of defensin and ixodidin did not change and ixodidin gene expression reduced after exposure of these cells to yeast. In the infection by A. marginale we detected defensin and ixodidin gene expression. Also, microplusin and defensin genes were silenced by RNA interference (RNAi) in A. marginale-infected BME26 cells, but we did not observe alteration in the number of MSP4 rickettsias

Made available in DSpace on 2015-03-26T13:47:27Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1156387 bytes, checksum: 78ed9fe351974511a2d1393bbd68273a (MD5) Previous issue date: 2003-02-28 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The immune response against the pathogen Anaplasma marginale was evaluated after the inoculation, in calves, with the strain AUFV1 2nd passage. For this, routine coloration techniques and immunohistochemistry techniques were performed in slides of superficial limph nodes. In the first week post inoculation, a proliferative response was observed in the paracortical areas and, in the third week, a great number of germinal centers. With the TUNEL technique, many cells in apoptosis were observed, in a number statistically significant six and 13 days post inoculation. Antigens of A. marginale presented by dendritic cells were detected with the Indirect Immunoperoxidase technique six days post inoculation, and these were not observed in the limph node of the negative control animal. The same technique was also performed with the purpose of identifying the surface antigens CD4, CD8 and WC1, and a little augment was observed only on the population of lymphocytes CD4+. Another experiment evolved the isolation of peripheral blood mononuclear cells (PBMCs), wich were isolated in the same days the limph nodes were coleted, for studies of proliferation and production of cytokines after reestimulation ex vivo. The mitogen Con A and initial bodies of A. marginale were utilized as positive controls. The PBMCs were estimulated wiht two synthetic peptides based on the structure of the surface protein of A. marginale MSP-2, named 13590 and 13591. A negative control was also utilized, were was added incomplete medium only. On the proliferative tests, only six days post inoculation the peptides were better than the negative control, with numbers statistically significants (p<0,05). On this way, we could conclude that the inoculated animals developed an adaptative immune response against A. marginale, probably evolving T helper cells. In relation to the synthetic peptides tested, we can only afirm that they are good candidates for the development of a vaccine against bovine anaplasmosis, but more studies have to be performed in this area. / Foi avaliada a resposta imune contra o patógeno Anaplasma marginale através da inoculação, em bezerros, da amostra AUFV1 2a passagem. Para tanto, foram realizadas técnicas de coloração de rotina e técnicas imunohistoquímicas em cortes de linfonodos superficiais. Inicialmente, foi observada uma resposta proliferativa na área paracortical e, a partir da terceira semana pós-inoculação, grande reatividade de centros germinais. Através da técnica de TUNEL, foram observadas inúmeras células em apoptose, em número estatisticamente significativo aos seis e 13 dias pós-inoculação. Antígenos de A. marginale apresentados por células dendríticas foram detectados pela técnica da Imunoperoxidase Indireta já aos seis dias pós-inoculação, não sendo observados no linfonodo do animal controle negativo. A técnica da Imunoperoxidase Indireta também foi utilizada para a detecção de antígenos CD4, CD8 e WC1, sendo observado um pequeno aumento apenas de linfócitos CD4+. Paralelamente a estes estudos, foram isoladas células mononucleares de sangue periférico (PBMCs), nos mesmos dias em que eram feitas as coletas de linfonodos, para estudos de proliferação e de produção de citocinas após reestimulação ex vivo. Como controle positivo, foi utilizado o mitógeno celular Concanavalina A, além de corpúsculos iniciais de A. marginale. As PBMCs foram estimuladas com dois peptídeos sintéticos baseados na estrutura da proteína de superfície de A. marginale MSP-2, os peptídeos 13590 e 13591. Também foi feito um controle negativo, onde era adicionado apenas meio de cultivo incompleto. Nos testes proliferativos, apenas aos seis dias pós-inoculação os peptídeos tiveram desempenho melhor, estatisticamente significativo (p<0,05), com relação ao controle negativo. Dessa forma, pode-se concluir que os animais inoculados desenvolveram uma resposta imune adaptativa contra o A. marginale, provavelmente envolvendo células T auxiliares. Quanto aos peptídeos sintéticos testados, serão necessários mais estudos, podendo-se afirmar apenas que eles são candidatos ao desenvolvimento de uma vacina eficaz contra o A. marginale.

Obligate intracellular pathogens must acquire host cell-derived nutrients to facilitate their survival. One such bacterial pathogen, Anaplasma phagocytophilum, replicates within neutrophils and non-phagocytic cells in a bacterial-modified, host cell-derived vacuole. The bacterium exploits host cell vesicular trafficking pathways to route nutrients to its vacuole and utilizes Rab GTPases, guanine nucleotide-dependent, vesicular trafficking regulators, to do so. We previously discovered that the A. phagocytophilum vacuolar membrane is decorated with a specific subset of Rab GTPases - Rab1, Rab4A, Rab10, Rab11, Rab14, Rab22A and Rab35. Rab1 is exclusively found on the endoplasmic reticulum (ER) and thus its localization suggests that the bacterium intercepts the ER. Rab10, which is found on the ER, trans-Golgi and recycling endosomes, localizes to the vacuolar membrane in a guanine nucleotide-independent and bacterial protein synthesis-dependent manner. This suggests that a bacterial-encoded protein is binding to and recruiting Rab10. In this study, we determined that A. phagocytophilum hijacks two very nutrient-rich sources in the secretory pathway - trans-Golgi- and endoplasmic reticulum-derived vesicles. A. phagocytophilum localizes perinuclearly adjacent to the Golgi apparatus during infection. A. phagocytophilum and Anaplasma marginale, an intravacuolar bovine pathogen, also localize near the smooth ER and rough ER in both mammalian and tick host cells. These results are supported by transmission electron microscopy analyses of infected cells. Membrane markers for the rough ER label the peripheries of A. marginale and A. phagocytophilum organisms in both mammalian and tick host cells, which suggests that they are translocated into the pathogen vacuole. Furthermore, membrane markers for trans-Golgi-derived vesicles, including endogenous Rab10, label the periphery of intravacuolar A. phagocytophilum organisms. Markers for the trans-Golgi and the ER co-fractionate with A. phagocytophilum in density gradient centrifugation studies. siRNA knockdown of Rab10 pronouncedly reduces delivery of trans-Golgi markers into the pathogen-occupied vacuole, significantly reduces infection, and impedes bacterial conversion to the bacterium’s dense-cored form. These results suggest that trans-Golgi recruitment is Rab10 dependent and is critical for bacterial development. We identified an outer membrane A. phagocytophilum moonlighting protein, uridine monophosphate kinase that specifically binds GST-Rab10 in affinity chromatography assays and interacts with Rab10 in vivo. We hypothesize that this surface protein is mediating the interaction of the bacteria with intravacuolar trans-Golgi derived vesicles. This interaction could be critical for the delivery of essential nutrients. Taken together, these data suggest that nutritional virulence mechanisms of A. phagocytophilum and A. marginale target the host secretory pathway. Additionally, they suggest a novel mechanism whereby pathogens translocate nutrient rich vesicles into the pathogen vacuole, thus delivering essential nutrients right to their front door.

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to cause the emerging tick-transmitted disease, human granulocytic anaplasmosis (HGA). Following entry, the pathogen replicates within a host cell-derived vacuole that fails to mature along the endocytic pathway, does not acidify, and does not fuse with lysosomes. Selective fusogenicity is prototypical of many vacuole-adapted pathogens and has been attributed, at least in part, to pathogen modification of the vacuolar inclusion membrane and/or to selective recruitment or exclusion of host trafficking regulators. As a result, the A. phagocytophilum-occupied vacuolar membrane (AVM) provides a unique interface to study the host-pathogen interactions critical to A. phagocytophilum intracellular survival. Diverse vacuole-adapted pathogens; including Chlamydia, Legionella, and Salmonella; selectively recruit host Rab GTPases to their vacuolar membranes to establish replicative permissive niches within their host cells. Rab GTPases coordinate many aspects of endocytic and exocytic cargo delivery. We determined that the A. phagocytophilum-occupied vacuole (ApV) selectively recruits a subset of fluorescently-tagged Rabs that are predominantly associated with recycling endosomes. Another emerging theme among vacuole-adapted pathogens is the ability to hijack ubiquitin machinery to modulate host cellular processes. Mono- and polyubiquitination differentially dictate the subcellular localization, activity, and fate of protein substrates. Monoubiquitination directs membrane traffic from the plasma membrane to the endosome and has been shown to promote autophagy. We show that monoubiquitinated proteins decorate the AVM during infection of promyelocytic HL-60 cells, endothelial RF/6A cells, and to a lesser extent, embryonic tick ISE6 cells. Importantly, tetracycline treatment concomitantly promotes loss of the recycling endosome-associated GFP-Rabs and ubiquitinated proteins and acquisition of the late endosomal marker, Rab7, and lysosomal marker, LAMP-1, implicating bacterial-derived proteins in the ApV's altered fusogenicity. Therefore, we rationalized that A. phagocytophilum-encoded proteins that associate with the AVM may establish interactions with the host cell that are important for intracellular survival. By focusing on A. phagocytophilum proteins that are induced during host infection, we identified the first two bacterial-encoded proteins -- APH_1387 and APH_0032 -- that modify the AVM. Although functional studies are hindered by the lack of a system to genetically manipulate Anaplasma, the pathobiological roles of APH_1387 and APH_0032 are likely unique, as both proteins exhibit very little or no homology with any previously described protein. APH_1387 and APH_0032 are present at the cytoplasmic face of the AVM, therefore they likely interact with host proteins. We demonstrate that ectopic expression of APH_1387 and APH_0032 inhibits the ApV development in A. phagocytophilum infected cells. The results presented in this dissertation contribute to our understanding of how A. phagocytophilum modifies the vacuolar membrane in which it resides to establish a safe haven and evade lysosomal degradation.

Submitted by Aline Batista (alinehb.ufpel@gmail.com) on 2017-04-13T19:13:08Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Hermann Eduardo Gonzáles Grau.pdf: 650172 bytes, checksum: 32cc0d96230e42711032018d51a55f04 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-04-13T19:42:46Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Hermann Eduardo Gonzáles Grau.pdf: 650172 bytes, checksum: 32cc0d96230e42711032018d51a55f04 (MD5) / Made available in DSpace on 2017-04-13T19:42:47Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Hermann Eduardo Gonzáles Grau.pdf: 650172 bytes, checksum: 32cc0d96230e42711032018d51a55f04 (MD5) Previous issue date: 2006-02-18 / Sem bolsa / Foi estudada a transmissão transplacentária do Anaplasma marginale em terneiros ao nascimento, produtos de vacas cronicamente infectadas, sem histórico de anaplasmose aguda durante a gestação. O estudo foi realizado em 30 terneiros e suas matrizes, no Município de Capão do Leão, região sul do Rio Grande do Sul, área de instabilidade enzoótica para A. marginale, onde as condições climáticas são desfavoráveis ao desenvolvimento dos vetores durante o período de inverno, quando as temperaturas médias são inferiores a 15ºC. Foram colhidas amostras de sangue dos terneiros antes da ingestão do colostro, aos 3 a 5 dias de vida, e das matrizes logo após o parto. Os testes sorológicos usados foram Imunofluorescência Indireta (RIFI) e Ensaio de Imunoadsorção Enzimática Indireto (ELISA I), e o exame direto foi através de Reação da Polimerase em Cadeia (PCR). Em 63,3% das matrizes, foi diagnosticada a infecção por A. marginale no PCR, enquanto que 100% das mesmas foram sorologicamente positivas na RIFI e 97% no ELISA. A transmissão transplacentária do agente foi constatada através da presença de anticorpos (IgG) em 10 % dos terneiros nascidos de mães positivas, e do DNA do agente em 10,5 % dos mesmos. Foi constatada uma concordância de 93,3% entre os resultados das técnicas sorológicas usadas, sendo que a precisão em relação ao PCR, foi de 80% para RIFI e de 76,7% para ELISA. São discutidos os resultados dos testes e a importância epidemiológica da transmissão transplacentária do A. marginale. / The transplacentary transmission of Anaplasma marginale was study in calves at the birth, from mothers cronically infected, without medical history of acute anaplamosis during the pregnancy. The study was carried out using 30 calves and their mothers, from Capão do Leão county, in the southern region of Rio Grande do Sul state, Brazil, which represents an area of enzootic instability to A.marginale infection, where during the winter season, the climatic conditions are hostile to the development of the vectors. The blood samples of the calves were collected before the colostrum ingest, from 3 to 5 days of live, and the mothers were also collected. The serological tests used were Indirect Fluorescent Antibody Technique (IFAT) and Indirect ELISA; the direct examination was carried out by Polymerase Chain Reaction (PCR). The results presented in the mothers were 63,3% in the direct examination by PCR test and in the indirect serological tests, IFAT and Indirect ELISA the percentile of positive, were respectively, 100% and 97%, to the infection by A.marginale. The transplacentary transmission of the agent was determined by the presence of antibodies (IgG) in 10% of the calves born of positive mothers, and by the agent DNA in 10,5% of them. An agreement of 93,3% was observed between the results of the serological techniques used, the precision in relation to the PCR test was 80% to IFAT and 76,7% to Indirect ELISA. The results of the tests and epidemiological importance of the transplacentary transmission of the A.marginale, are discussed.

Made available in DSpace on 2016-04-28T20:16:32Z (GMT). No. of bitstreams: 1 2006-Valeria Regia Franco Sousa.pdf: 1545960 bytes, checksum: 07990d273ccfe1ac6c38929e5d3d100c (MD5) Previous issue date: 2006-02-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / This work had for objective to investigate the infection for Ehrlichia canis and Anaplasma platys in the dogs taken care of in the Veterinarian Hospital of the Universidade Federal de Mato Grosso, through the examination of blood smears and of the PCR, analyzing the clinical and laboratories findings. During the period of May of 2004 the July of 2005, 195 dogs with suggestive clinical signs of ehrlichiosis or had contact with the infection had been examined. The canine cyclic thrombocytopenia, caused for the A. platys, provokes reduction of platelets to each 14 days, without provoking severe signs, but when associated to the infection for E. canis the clinical gravity it increases. In the thirteen dogs taken care of with positive PCR for E. canis diagnosised by the PCR it was possible to verify the diversity of signals, with statistical significant predominance of the hemorrhagic riots. Already in the 195 dogs tested for the examination of blood smears it did not have predominance of no clinical signs. The hematologic findings had also been nonspecific, occurring in such a way anemia, leukopenia and thrombocytopenia, how much normality or increase of the cells. The alterations observed in the analysis biochemist had not been exclusive of the groups with infection. In these groups increase of plasma proteins occurred, with hyperglobulinaemia, without, however to have significant difference, despite this finding being frequent in ehrlichiosis. From the analysis of the PCR it was confirmed that the infection for E. canis and A. platys occur in the dogs taken care of in the Veterinarian Hospital of the UFMT, having to be taken writ of prevention, mainly in the control of the vector, since all the ticks found in the examined dogs had been of species Rhipicephalus sanguineus. / Este trabalho teve por objetivo investigar a infec??o por Ehrlichia canis e Anaplasma platys nos c?es atendidos no Hospital Veterin?rio da Universidade Federal de Mato Grosso, atrav?s das t?cnicas de esfrega?o sang??neo e de PCR, analisando os achados cl?nicos e laboratoriais. Durante o per?odo de maio de 2004 a julho de 2005 foram examinados 195 c?es com sinais cl?nicos sugestivos de erlichiose ou com contactantes com a infec??o. A trombocitopenia canina c?clica, causada pelo A. platys, provoca diminui??o das plaquetas a cada 14 dias, sem provocar sinais severos, mas quando associada ? infec??o por E. canis a gravidade cl?nica aumenta. Nos treze c?es atendidos com PCR positiva para E. canis foi poss?vel verificar a diversidade de sinais, com predomin?ncia estatisticamente significativa dos dist?rbios hemorr?gicos. J? nos 195 c?es testados pela t?cnica de esfrega?o sang??neo n?o houve predom?nio de nenhum sinal cl?nico. Os achados hematol?gicos tamb?m foram inespec?ficos, ocorrendo tanto anemia, leucopenia e trombocitopenia, quanto normalidade ou aumento das c?lulas. As altera??es observadas na an?lise bioqu?mica n?o foram exclusivas dos grupos com infec??o. Nestes grupos ocorreu aumento das prote?nas plasm?ticas, com hiperglobulinemia, sem, no entanto haver diferen?a significativa, apesar deste achado ser freq?ente na erliquiose. A partir da an?lise da PCR confirmou-se que a infec??o por E. canis e A. platys ocorre nos c?es atendidos no Hospital Veterin?rio da UFMT, devendo ser tomadas medidas preventivas, principalmente no controle do vetor, j? que todos os carrapatos encontrados nos c?es examinados foram da esp?cie Rhipicephalus sanguineus.

Obligate intracellular pathogens must invade host cells to survive and pose a global health risk. As such, internalization is a critical life stage and represents an excellent therapeutic target. Oxidoreductase exploitation is a thematic invasion strategy among obligate intracellular pathogens. Delineating the mechanisms and proteins mediating this exploitation could identify novel therapeutic targets for many important pathogens. Anaplasma phagocytophilum infects neutrophils by an incompletely defined mechanism, resulting in the emerging potentially fatal disease, human granulocytic anaplasmosis. The bacterial adhesin, Asp14, contributes to invasion by virtue of its C-terminus engaging an unknown receptor. Yeast two-hybrid analysis identified protein disulfide isomerase (PDI) as a putative Asp14 binding partner. Co-immunoprecipitation confirmed this interaction and identified the Asp14 C-terminus as critical to it. PDI reductase activity inhibition impaired bacterial infection of, but not binding to, host cells. A. phagocytophilum failed to productively infect myeloid-specific PDI conditional knock-out mice. This is the first demonstration of microbial PDI exploitation in vivo. Infection of PDI inhibited cells was rescued when bacterial, but not host surfaces were reduced with the reducing agent tris(2-carboxyethyl)phosphine (TCEP). Furthermore, TCEP restored bacterial infectivity after Asp14 inhibition using an antibody that reduces infection. Mutational analyses identified Asp14 residues critical for binding PDI. These data demonstrate that Asp14 binds and brings PDI to disulfide bonds within A. phagocytophilum surface protein(s) that it reduces, enabling infection. Targeting the Asp14 C-terminus could benefit approaches to prevent/treat granulocytic anaplasmosis. A similar approach would identify proteins from other obligate intracellular pathogens that could prove to be protective targets.

Thesis (MSc (Veterinary Tropical Diseases)--University of Pretoria, 2009. / Includes bibliographical references. Also available in print format.

Bovine anaplasmosis is caused by Anaplasma marginale and A. centrale. The most pathogenic and important species for cattle production is A. marginale, and is widely distributed in tropical, subtropical and temperate regions of the world. A. marginale is an intra-erythrocyte rickettsia of susceptible ruminants, biological and mechanically transmitted by ticks and hematophagous insects. The tick Rhipicephalus (Boophilus) microplus is the main vector of A. marginale in Brazil. The congenital form of transmission in cattle may occur, causing the neonatal anaplasmosis. The outer membrane of A. marginale includes six well characterized major surface proteins, MSP1a, MSP1b, MSP2, MSP3, MSP4 and MSP5, which play important role in the development of the immune response of infected animals. In this study, we have used the Phage Display technology to identify specific peptides that were immunoreactive to monoclonal antibodies anti-A. marginale proteins. Peptide selection was performed using a subtractive selection of a peptide library with 12 random amino acids, Ph.D.-12, expressed on the surface of the M13 filamentous phage concurrently against the anti-MSP1a and anti-MSP2. After four rounds of selection and validation by ELISA, the selected peptides have recognized only the anti-MSP1. Analysis of bioinformatics identified 45 peptides, which showed the protein consensus sequence STxS that was represented in 78% of selected phages. Due to the multiple motif repeats found in MSP1 protein, the STSSxL motif may become an important biological target, with potential use in diagnostic tests and vaccine for the control of Anaplasma marginale. / Anaplasmose bovina é uma infecção causada por Anaplasma marginale e A. centrale. A espécie mais patogênica e de maior importância para bovinos é a A. marginale e está amplamente distribuída nas regiões tropicais, subtropicais e temperada do mundo. A. marginale é uma rickettsia intraeritrocitária de ruminantes susceptíveis, transmitido biológica e mecanicamente por carrapatos e insetos hematófagos. O carrapato Rhipicephalus (Boophilus) microplus é o principal transmissor de A. marginale no Brasil. A forma congênita de transmissão em bovinos pode ocorrer, ocasionando a anaplasmose neonatal. A membrana externa do A. marginale inclui seis proteínas principais de superfície (MSPs) bem caracterizadas, designadas de MSP1a, MSP1b, MSP2, MSP3, MSP4 e MSP5 e desempenham papel importante no desenvolvimento da resposta imune de animais infectados. Para o desenvolvimento deste estudo, foi utilizada a técnica de Phage Display para identificar peptídeos ligantes a anticorpos monoclonais reativos a proteínas de Anaplasma marginale a partir de bibliotecas de peptídeos recombinantes por meio da tecnologia Phage Display. Para seleção dos peptídeos foi realizado uma seleção subtrativa utilizando uma biblioteca de peptídeos com 12 aminoácidos randômicos, Ph.D.-12, expressa na superfície do fago filamentoso M13 concomitantemente contra os anticorpos anti-MSP1a e anti-MSP2. Após quatro ciclos de seleção e validação por ELISA, o conjunto de peptídeos selecionados apresentou ser unicamente reconhecido pelo anticorpo anti- MSP1. Análises de bioinformática identificaram 45 peptídeos, que apresentaram o motivo proteico consenso STxS, representado em 78% dos fagos seqüenciados. Devido aos múltiplos sítios repetidos encontrados na proteína MSP1, o motivo proteíco STSSxL pode ser um importante alvo biológico, com potencial utilização em ensaios diagnósticos e vacinais para o controle de Anaplasma marginale. / Mestre em Genética e Bioquímica

During a period of 3 years, from March to July 2014, 2015 and 2016, ticks were collected from dogs and cats in shelter facilities for abandon animals in Česke Budejovice, South Bohemia. In total, 343 ticks were found on 106 pets: 67 domestic dogs and 39 cats. All collected ticks, that were identified as Ixodes ricinus and Ixodes hexagonus, were tested for the presence of spirochetes from Borrelia burgdorferi sensu lato (s.l.) complex, Rickettsia spp., Anaplasma spp, and Babesia spp using conventional PCR and nested PCR. Identification of pathogens was done by following sequencing of amplicons. Out of all tested ticks, 49,56% were proved to be infected at least with one pathogen. Co-infection of at least two different pathogens was determined in 18 ticks (5,2%). The aim of the present study was to estimate the role of accompanying animals (cats and dogs) in the circulation of ticks and tick-borne pathogens, to determine the frequency of pathogenic infections in dog and cat associated ticks, to evaluate the current risk of infection for dogs and cats, with respect torisk forhumans living in the area of České Budějovice.